Insight is provided into the mechanisms underlying α-synuclein turnover and suggests promising avenues for disease-modifying interventions in synucleinopathies.
Abstract
Insufficient understanding of α-synuclein turnover mechanisms has impeded successful clinical translation for Parkinson's disease (PD). Here, we pinpointed cholesterol 25-hydroxylase (CH25H) as a pivotal regulator of α-synuclein degradation. Through bulk RNA sequencing of substantia nigra tissue from the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, along with reanalysis of published datasets from induced pluripotent stem cell-derived astrocytes of patients with PD, we observed an elevated CH25H expression in PD-associated astrocytes. This finding was validated by combined fluorescence in situ hybridization for Ch25h and immunofluorescence staining for GFAP in mouse substantia nigra sections. Conditional knockout or knockdown of astrocytic Ch25h alleviated PD-like motor deficits and reduced dopaminergic neuronal loss in MPTP and α-synuclein preformed fibril (PFF) mouse models. Using 4D label-free proteomics and molecular docking approaches, we uncovered a shared binding domain on p62 where both CH25H and α-synuclein interact. Proximity ligation assays in cultured astrocytes showed that Ch25h overexpression promoted formation of p62/CH25H complex, whereas it inhibited p62/α-synuclein interaction. Conversely, Ch25h knockdown enhanced p62/α-synuclein complex formation and facilitated α-synuclein degradation. 25-Hydroxycholesterol, the enzymatic by-product of CH25H, did not affect the expression of α-synuclein in astrocytes, suggesting an activity-independent influence of CH25H on α-synuclein clearance. In addition, treatment with a p62 polypeptide (60 to 90 amino acids) effectively facilitated α-synuclein clearance by sequestering free CH25H in both cultured astrocytes and mice in the PFF model. Collectively, our study provides insights into the mechanisms underlying α-synuclein turnover and suggests promising avenues for disease-modifying interventions in synucleinopathies.
Background Post-translational modifications, particularly SUMOylation, plays a crucial role in α-synuclein (α-syn) aggregation, a key pathological feature of Parkinson’s disease (PD). Curcumin, a natural polyphenol, has shown neuroprotective potential, but its effects on SUMOylation-related signaling in PD remain unclear. Objective This study aimed to investigate whether curcumin modulates α-syn SUMOylation and to elucidate the underlying molecular mechanisms in PD model mice. Methods A PD model was established in male C57BL/6 mice via unilateral intrastriatal injection of α-syn preformed fibrils (PFFs). Six months after α-syn PFFs injection, mice were treated intravenously with curcumin (25 mg/kg/day) or vehicle for 1 month. Behavioral tests (open field, rotarod) assessed motor function. Neuropathology was evaluated by immunohistochemistry and western blotting for tyrosine hydroxylase (TH), phosphorylated α-syn (p-syn), SUMOylation pathway components (SUMO1, SAE2, UBC9, PIAS1/2), and ubiquitin. Striatal dopamine levels were measured by HPLC. Results Curcumin treatment ameliorated motor deficits and anxiety-like behaviors in PD mice. It partially preserved dopaminergic neurons and reduced p-syn aggregation in the substantia nigra, accompanied by increased striatal dopamine levels. Mechanistically, curcumin was associated with reduced SUMO1 and increased ubiquitin levels, suggesting modulation of SUMOylation-related signaling. Among SUMOylation enzymes, UBC9 expression was decreased, whereas E1 (SAE2) and E3 (PIAS1/2) components were not substantially affected. Conclusion Our findings demonstrated that curcumin exerted neuroprotective effects in a PD model by attenuating α-syn pathology. The protective mechanism involves the inhibition of α-syn SUMOylation, primarily through the downregulation of the UBC9 enzyme. This study identifies UBC9-mediated SUMOylation as a potential target for curcumin and highlight a promising strategy for modifying α-syn-associated pathology in PD.
Yao Li, Zhuang Zhu, Kezhong Zhang· Frontiers in Neuroscience· 0 citations
OBJECTIVE
To investigate the effects of targeting DYNC1I1 in cellular and mouse models of Parkinson's disease (PD). Methods The scRNA-seq dataset GSE243639 was analyzed to prioritize autophagy- and transport-related candidate genes in post-mortem substantia nigra samples from patients with PD and controls. SH-SY5Y cells were treated with 1mM MPP⁺ for 48h, and male C57BL/6 mice received MPTP at 30mg/kg/day for 5 consecutive days. DYNC1I1 was silenced with siRNA in cells and targeted with AAV-DYNC1I1 in mice. Autophagic-lysosomal status was evaluated using tandem mCherry-GFP-LC3B fluorescence and autophagy- and lysosome-related markers. Motor performance was assessed using the pole and rotarod tests.
RESULTS
DYNC1I1 was prioritized as a candidate gene on the basis of PD-associated differential expression, overlap with autophagy-related genes, enrichment in a dopaminergic neuron-related subpopulation, and biological relevance to intracellular transport. In MPP⁺-treated cells, DYNC1I1 knockdown reduced α-synuclein accumulation and phosphorylation and improved autophagic-lysosomal marker profiles. In MPTP-treated mice, AAV-DYNC1I1 treatment improved motor performance and TH-positive signal and reduced α-synuclein pathology and autophagic-lysosomal impairment. AAV-DYNC1I1 treatment was also associated with higher p-PI3K/PI3K and p-AKT/AKT ratios.
CONCLUSIONS
DYNC1I1-targeting interventions attenuated PD-related phenotypes in cellular and mouse models. The observed effects were accompanied by improved autophagic-lysosomal marker profiles and increased PI3K/AKT phosphorylation.
Yuyu Li, Li Xu, Hui Sun et al.· Brain Research Bulletin· 0 citations
Understanding how preclinical models of Parkinson’s disease (PD) reflect mechanisms underlying PD is crucial for successful drug development. To illuminate the translational value of two well-established rat synucleinopathy models – substantia nigral AAV-delivered human mutant α-Synuclein (α-Syn-hA53T) and striatal α-Syn preformed fibril (PFF)-injection – unbiased transcriptomic and proteomic analyses were employed and the genes, proteins, and pathways disrupted in striatum and substantia nigra were assessed. Unbiased analyses identified 388 proteins altered in hA53T-α-Syn and 1550 in PFF-α-Syn compared to control brain. Pathway and correlation analyses of the altered proteins revealed distinct processes impacted in each model: Dopaminergic signaling/metabolism, mitochondria and energy metabolism, and motor processes were most predominately disrupted in the AAV-hA53T-α-Syn model, whereas immune response, intracellular/secretory and synaptic vesicles, and autophagy were predominately disrupted in PFF-α-Syn model. Both models reflected aberrant synaptic function, neural growth/remodeling, and protein localization. The set of disrupted proteins/genes were compared to that aberrantly expressed in human PD and dementia with Lewy bodies (DLB) and candidate drug targets in a “PD drug development pipeline”, revealing potential disease biomarkers and optimal models for testing novel therapeutics. Not applicable.
Britney N. Lizama, Rick Shin, Hilary A. North et al.· Discover Neuroscience· 0 citations
Overexpression of α-synuclein (α-syn), an inherently disordered protein, triggers chronic activation of the mitochondrial unfolded protein response (UPRmt) pathway in Caenorhabditis elegans with enhanced dopaminergic (DAergic) neurodegeneration. Introduction of a loss-of-function(lf) mutation in atfs-1, the main transcriptional regulator of the UPRmt, into α-syn nematodes results in significant neuroprotection from α-syn-induced DA neuron loss. Using this sensitized neuroprotective background, we performed a F3 forward genetic screen in C. elegans atfs-1(lf) mutants to identify molecular components associated with the modulation of neurodegeneration in α-syn-expressing DA neurons. Homozygous mutant animals were examined for enhanced neurodegeneration; multiple independent alleles were uncovered. Among these, we identified new nonsense alleles encoding the histone lysine demethylases (H3K27me3), jmjd-1.2 (orthologous to human KDM7A, PHF2, and PHF8) and jmjd-3.1 (homologous to yeast CYC8). Another line carried a nonsense allele of twk-14. This gene encodes a conserved protein termed KCNK12 in mammals that facilitates passive background K+ leak currents to set and stabilize resting membrane potential. To further examine the association of these gene products in DA neurodegeneration, we used neuron-specific RNA interference, mutants, or both. DA neurodegeneration was observed in the α-syn + atfs-1(lf) background when jmjd-1.2, jmjd-3.1, or twk-14 were individually depleted. These results provide evidence that jmjd-1.2 and jmjd-3.1, which encode previously characterized H3K27me3 demethylases, and the uncharacterized twk-14 gene product, orthologous to human KCNK12, naturally confer protection from α-syn-induced neurotoxicity.
Karolina Willicott, Joy D. Iroegbu, Madeline R. Greene et al.· bioRxiv· 0 citations
BACKGROUND
Parkinson's disease (PD) is a prevalent neurodegenerative disorder hallmarked by progressive loss of midbrain dopaminergic (DA) neurons, neurite atrophy, neurotransmitter imbalance, and motor dysfunction. Therapeutic strategies targeting neuronal integrity and neuroinflammation hold translational value beyond conventional symptomatic therapies for PD. Sarsasapogenin, a natural steroidal saponin, exhibits documented neuroprotective properties in multiple neurological diseases. However, its therapeutic potential and underlying mechanisms in PD remain to be elucidated.
METHODS
The neuroprotective efficacy of sarsasapogenin was assessed in a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse PD model and 1-methyl-4-phenylpyridinium ion (MPP+)-insulted primary cortical and dopaminergic neuronal cultures. Motor function was evaluated via a variety of behavioral tests. Survival of dopaminergic neurons was quantified using tyrosine hydroxylase (TH) immunostaining. Levels of monoamine neurotransmitters and their metabolites were measured using high-performance liquid chromatography (HPLC), while serum proinflammatory cytokines were determined using enzyme-linked immunosorbent assay (ELISA). Neurite outgrowth and neuronal viability were examined under both preventive and therapeutic treatment regimens in vitro. Potential molecular targets of sarsasapogenin were screened and identified via Drug Affinity Responsive Target Stability (DARTS) coupled with molecular docking.
RESULTS
Twenty-one-day sarsasapogenin intervention mitigated aberrant motor deficits in MPTP-treated mice, rescued TH+ dopaminergic neurons in the substantia nigra, restored homeostasis of dopamine and norepinephrine, normalized the turnover of monoamines, and decreased peripheral levels of the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). In primary neuronal cultures, sarsasapogenin significantly ameliorated MPP+-triggered neurite atrophy and neuronal loss in both cortical and ventral mesencephalic dopaminergic neurons. DARTS screening combined with molecular docking identified serpin family H member 1 (Serpin) H1 as a candidate binding protein of sarsasapogenin, with a favorable binding affinity of -8.6 kcal/mol.
CONCLUSIONS
Sarsasapogenin exerts comprehensive neuroprotective effects in preclinical PD models via synergistic modulation of dopaminergic neuronal survival, neurotransmitter balance, inflammatory inhibition, and neurite outgrowth. These findings highlight sarsasapogenin as a promising multi-target candidate for PD intervention, whereas the precise functional role of Serpin H1 in mediating its neuroprotective actions warrants further in-depth investigation.
Wendi Deng, Jinfeng Hong, Weixia Li et al.· Journal of Integrative Neuro...· 0 citations
Accumulation of misfolded α-synuclein protein in intracellular inclusion bodies of dopaminergic neurons underlies the pathogenesis of Synucleinopathies, which include Parkinson’s Disease (PD) and Dementia with Lewy Bodies (DLB). Therefore, preventing the accumulation of misfolded α-synuclein in dopaminergic neurons or their clearance could in principle offer intervention strategies against Synucleinopathies, which currently remain untreatable. In this study, we employ the Affinity-directed PROtein Missile (AdPROM) system consisting of the substrate receptor of the CUL2-E3 ligase complex VHL and a nanobody selectively recognising the human α-synuclein protein and demonstrate targeted degradation of endogenous α-synuclein from human cell lines with remarkable selectivity. We further demonstrate that targeted degradation of α-synuclein impedes the pre-formed fibril (PFF)-induced aggregation of α-synuclein in primary neurons derived from rats expressing human α-synuclein. This approach also represents the first demonstration of nanobody-guided proteasomal degradation of some reported clinically relevant α-synuclein variants.
Bill Carton, Géraldine Gelders, Gajanan Sathe et al.· npj Parkinson's Disease· 0 citations